Techniques for the study of the cell biology of Entamoeba histolytica in axenic culture have been developed and exploited. 1. Conditions for growing E. histolytica as colonies from single cells in agar were determined. Colony-forming efficiency was determined by the strain, age of cells, type, concentration, and method of solififying the agar. Eleven of 12 E. histolytica strains and all 6 reptilian strains (3 species) tested formed colonies in agar. Clones which were isolated by this easy and non-selective method did not differ from the parental population in growth characteristics or virulence for newborn hamster liver. This method is also useful for quantitation of viability as in drug testing. 2. Other studies have concentrated on the physiological requirements for motility of E. histolytica trophozoites, including the effects of varying pH, redox potential, osmolarity, and divalent metal ions. These studies have led to the formulation of "Motility Buffer" a defined composition medium for the short term maintenance of viability (as shown by the agar method) and motility of E. histolytica in the absence of growth.